The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm2 and 5.74 μg protein/cm2 for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH−lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29−lysozyme, 0.95 log for EVOH 44−lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.